Cellular and molecular mechanisms involved in T cell-mediated autoimmunity against immunologically privileged retinal antigens are being studied. The questions are aimed at elucidating the natural development and maintenance of self-tolerance to retinal antigens, and defining the processes that lead to their pathological breakdown. The goal is to use this knowledge for designing novel and rational strategies for immunotherapy. The experimental approaches utilize the model of experimental autoimmune uveoretinitis (EAU), which resembles immune-mediated uveitic diseases in humans that can lead to blindness. EAU is induced in mice and rats by immunization with retinal antigens such as IRBP, Arrestin (S-Ag), or their component peptide epitopes, or by infusion of cultured lymphocytes that recognize these antigens. The mechanisms controlling disease susceptibility and pathogenesis are being defined at the genetic, developmental, and immunological levels. Novel approaches to disease regulation are devised based on these findings. We have previously shown that the threshold of susceptibility to EAU is in controlled in part by thymic derived "natural" CD4+CD25+ regulatory T cells (T-regs). We investigated the requirement for expression of the cognate Ag (IRBP) in the thymus to generate these cells. Our data indicate that thymic expression of IRBP is dispensable to generate T-regs that protect from EAU. The antigenic specificity of the T-regs generated in the absence of endogenous IRBP is being investigated. As an attempt to strengthen what we believe to be deficient peripheral tolerance to retinal antigens, we are studying therapeutic induction of tolerance in adult mice by hydrodynamic IV vaccination with a naked DNA plasmid encoding an IRBP fragment. This method requires only a single vaccination with 10 ?g of DNA. The major site of expression of the hydrodynamically injected protein is in the liver. It is highly effective in preventing disease, but only moderately effective in reversing disease elicited by already primed T cells. We are currently studying the cellular mechanisms leading to the protection. Another approach to induce peripheral tolerance is use of altered peptide ligands (APL) of the uveitogenic epitope that bind the MHC and/or TCR with reduced avidity. Two candidate APLs, that do not induce EAU when used in what would constitute a uveitogenic immunization regimen, but protect from EAU induced by the native epitope, were identified and are being studied. Innate immune responses to microbes, that create a pro-inflammatory environment, are thought to play a role as environmental triggers of autoimmunity. We are studying the dependence of EAU induction on signaling through the MyD88 receptor pathway using MyD88 KO mice, as well as TLR2, TLR 4, TLR 9, IL-18 and IL-1RI gene knockout mice, all of which signal through the MyD88 pathway. MyD88 deficient mice failed to develop EAU and made enhanced IL-4, IL-5 and IL-10 responses, but strongly reduced IL-6, IFN-gamma. and TNF-alpha, response to IRBP. (Interestingly, the "Th2" cytokines IL-4, IL-5 and IL-10 were produced by non-T cells, mostly B lymphocytes). Unexpectedly, all 3 TLR KOs and the IL-18 KO mice remained fully susceptible to EAU. In contrast, IL-1 KO mice were resistant. Thus, although the MyD88 pathway is necessary for EAU to be induced, TLR2, TLR 4, TLR 9 AND IL-18 signaling is either unnecessary or redundant. In contrast, the requirement for IL-1 signaling is necessary and nonredundant for EAU induction, and can by itself explain resistance of the MyD88 mice to EAU. Dendritic cells (DC) are important antigen presenting cells (APC) in induction of immune and autoimmune responses. We are developing an EAU model where disease is induced with in vitro-matured, antigen pulsed DC. We are able to obtain large numbers of splenic DC from mice injected hydrodynamically with Flt-3L and purify them to homogeneity. When matured (by culture with LPS and anti-CD40), pulsed with a uveitogenic IRBP peptide and injected into naive syngeneic mice, these DC induce vigorous immune responses and elicit typical EAU-like ocular inflammation. This model is an alternative to active immunization with uveitogenic proteins in complete Freund?s adjuvant and will permit to study various manipulations and agents for their ability to regulate activated DC capable of inducing EAU. In collaboration with the Oppenheim group at NCI we demonstrated that lymphocytes and immature dendritic cells exhibit chemotactic responses to the retinal antigens S-Ag and IRBP. The chemokine receptors CXCR5 and CXCR3 mediated the chemotactic effect of IRBP, while only CXCR3 was required for the S-Ag signal. We hypothesize that these responses may underlie attraction of immunocytes to sites of tissue damage and may thus facilitate processes of tissue repair and regeneration. When natural control mechanisms fail, or are subverted, such responses may lead to development of an adaptive response of the Th1 phenotype, leading to autoimmune tissue pathology. For a long time IL-12 was thought to be a necessary cytokine for induction of EAU and similar autoimmune diseases associated with a Th1 response. This was concluded on the basis of data obtained with monoclonal antibodies and knockout mice targeting the p40 chain of IL-12. However, recently another cytokine was discovered which shares the p40 chain with IL-12, and was shown to be involved in a number of autoimmune diseases. We are currently dissecting the roles of IL-12 and IL-23 in EAU using mice deficient in the non-shared receptors (p35 for IL-12 and p19 for IL-23), and the respective neutralizing monoclonal antibodies. Data indicate that IL-23, not IL-12, is an obligatory cytokine for induction of EAU. Ocular immune privilege, composed in part of inhibition of inflammatory processes in the eye, plays an important role in uveitis. We have previously shown that retinal glial Muller cells inhibit proliferation of T cells in an antigen-nonspecific fashion by a contact dependent mechanism. Using primary cultures of murine Muller cells, we have examined a large number of candidate molecules that might be involved in this phenomenon. Preliminary data identify thrombospondin-1, a molecule with many functions including activation of TGF-b, as partly responsible part of the suppressive effect of Muller cells. Studies are underway to examine the mechanism and identify additional molecules that might be involved in suppression. We have developed an antigen-MHC class II-Ig dimer, a specific reagent based on the major pathogenic peptide of IRBP. We showed that this reagent detects and modulates the function of a pathogenic T cell line that induces EAU. With the help of this reagent, we demonstrated that the major pathogenic epitope of IRBP for B10.RIII mice can be presented by both IA and IE molecules, possibly explaining the unusually high pathogenicity of this peptide. This reagent will permit direct study of antigen-specific pathogenic T cells in the EAU model and raises the possibility of using antigen-MHC multimers as research tools in clinical uveitis. Within the scope of our studies on genetic susceptibility to uveitis, we have defined several new genetic regions associated with susceptibility, that are shared with regions reported for other autoimmune and non-autoimmune diseases, among them experimental autoimmune encephalomyelitis, pristane induced arthritis and type 2 diabetes. This indicates that uveitis uses similar pathways as other autoimmune and inflammatory diseases, which may suggest use of common therapeutic approaches.